Separation of Neutrophils from Human Blood

Greg Denomme denomme at FHS.CSU.MCMASTER.CA
Mon Feb 28 13:44:22 EST 1994


Hello netters:

  If you want neutrophils try the following -

Add 5 mL of phosphate buffered saline to 5 mL of whole blood
Add 2 mL of 6% Dextran-T500 (in normal saline), mix well
  and let sit for 1 hour.
Harvest the white blood cells, approx. 8 mL (most of the red blood cells
settle out)
Underlay WBCs with 4 mL of Ficoll-Hypague and spin at 500 x g for 30 minutes
Neutrophils and the few RBCs found below the F-H
Remove supernatant and F-H.
Resuspend neutrophils/RBCs in PBS
Lyse red blood cells with H2O (if desired)

Hope this helps
Cheers,
Greg

Gregory Denomme, PhD
Department of Pathology
McMaster University
email: denomme at fhs.csu.mcmaster.ca


On 26 Feb 1994, Basavaraju Shankarappa wrote:

> Alex from lpss at unixhg.ubc.ca
> wrote:
> > 
> > As the title says, we are trying to separate neutrophiles from human
> > peripheral blood. Currently, we are using the Ficol-Hypaque gradient
> > centrifugation method. It's very time consuming. I'd to know other
> > methods, more efficient ones, such as: magnetic beads, column, etc. If
> > anyone know or used such methods, pleaselet me know.
> > Thanks
> > Alex
> 	I have seen a product advertised somewhere in literature
> called LEUKOPREP.  I think it was meant specifically to isolate 
> DNA from PBMC of blood.  I have no idea how this works or how 
> efficient is this.  
> 	If someone ever used this, would you please post an
> answer.  Now that many kits (Hi Jim 8-)) can be directly used to 
> extract DNA from the whole blood, I guess people have stopped using any
> labor intensive methods for this purpose.  
> Raj Shankarappa
> bsh at med.pitt.edu
> 
> 






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